The arterial wall and arterial valves are complex macromolecular structures. One of the major elements of these structures is the scaffold that provides the strength and flexibility to perform the task in hand either retaining the blood in vessels against the arterial pressure or maintaining pressure via the function of coronary valves. In the last several years it has become apparent that the actual microstructure and composition of these macromolecules could influence the progress of different disease states most notably atherosclerosis and valve calcification. To gain a better understanding of this process, we have embarked on studies to understand the fine structure of the macromolecules in arterial vascular bed using a novel optical imaging technique that relies on the non-linear excitation (NLE) of collagen and elastin to provide sub-micron images of their structure in unfixed fresh samples together with direct monitoring of water permeation through the wall using Coherent anti-Stokes Raman Scattering(CARS). Using these approaches we have made the following observations:1) We have adapted CARS microscopy to the evaluation of water movement in biological tissues using femtosecond excitation pulses, rather than the classical picosecond pulses, that delivers more energy as well as covers the bandwidth of the water raman scattering. This approach increases the signal to noise of the conventional approach by nearly 200 fold. 2) Using deuterium as a tracer we have established using CARS microscopy that the major barrier to water permeability, and thus where the largest pressure gradient is, is at the basolateral membrane of the endothelial cell. This region composed of the basolateral endothelial plasma membrane and the protein-rich basement membrane is apparently the major barrier to water and likely other solutes across the wall. Thought the endothelial cell has been believed to play a role as the water barrier, the specific membrane had not previously been determined. We are now characterizing the water channels in these membranes proposing that the endothelial apical cell membrane will be much richer in aquaporin channels than its less permeable basolateral membrane. In addition, we are determining the effect of different fixatives and detergents on the water permeability barrier, or the so called hyroseal of the arterial wall. 3) We have identified decorin and biglycan as the major binding sites for LDL in the valve leaflet and renal artery wall ostia. Based on the relative concentration of the interaction sites of decorin/biglycan and LDL, we have decided to target the LDL electrostatic binding sites to evaluate the interference of this interaction in the progression of atherosclerosis. Specific modification of the LDL lysine amino acid residues as well as model molecules such as Heparin sulfate has demonstrated that this strategy is feasible by significantly inhibiting LDL association with decorin in specially designed in vitro assays. One of the major issues is to find molecules with high affinity to the LDL macromolecule sites with minimum off target activity. 4) We have initiated studies in normal and atherosclerosis prone transgenic mice to correlate the development of the arterial macromolecular structures using NLE microscopy. We have completed the initial study that has characterized the development of the macromolecular structures in the normal mouse aorta and begun to follow the development of atherosclerosis in disease prone models. We have also recently demonstrated that CARS microscopy can directly observe the fatty acid C-H bonds this tissue. We will use CARS to evaluate the deposition of fatty acids and lipids in the vascular wall in the follow up study completing our minimally invasive approach for evaluation of the development of atherosclerotic disease in this animal model. This study when completed will provide an in depth analysis of the development of the macromolecular structures of the arterial wall in a mammalian model and permit the background for chronic studies with different strategies to reduce atherosclerosis.